The combined effects of Map3k1 mutation and dioxin on differentiation of keratinocytes derived from mouse embryonic stem cells

Epithelial development starts with stem cell commitment to ectoderm followed by differentiation to the basal keratinocytes. The basal keratinocytes, first committed in embryogenesis, constitute the basal layer of the epidermis. They have robust proliferation and differentiation potential and are responsible for epidermal expansion, maintenance and regeneration. We generated basal epithelial cells in vitro through differentiation of mouse embryonic stem cells (mESCs). Early on in differentiation, the expression of stem cell markers, Oct4 and Nanog, decreased sharply along with increased ectoderm marker keratin (Krt) 18. Later on, Krt 18 expression was subdued when cells displayed basal keratinocyte characteristics, including regular polygonal shape, adherent and tight junctions and Krt 14 expression. These cells additionally expressed abundant Sca-1, Krt15 and p63, suggesting epidermal progenitor characteristics. Using Map3k1 mutant mESCs and environmental dioxin, we examined the gene and environment effects on differentiation. Neither Map3k1 mutation nor dioxin altered mESC differentiation to ectoderm and basal keratinocytes, but they, individually and in combination, potentiated Krt 1 expression and basal to spinous differentiation. Similar gene-environment effects were observed in vivo where dioxin exposure increased Krt 1 more substantially in the epithelium of Map3k1+/- than wild type embryos. Thus, the in vitro model of epithelial differentiation can be used to investigate the effects of genetic and environmental factors on epidermal development.

The dioxin-like chemicals (DLCs) represent a large group of chemicals that are wide-spread environmental contaminants 18 . The DLCs are generated either naturally through processes like forest fires and volcanic eruptions or by industrial activities, such as incomplete combustions. These chemicals are stable in the environment with a half-life of several years. Therefore, human exposure is inevitable. DLC exposure has been linked to many developmental defects [19][20][21][22] . Using 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) as a model DLC congener, studies in laboratory rodents show that in utero dioxin exposure causes diverse developmental abnormalities, including, but not limited to, hydronephrosis, cleft palate, and vaginal thread formation 23 . In the epidermis, dioxin is shown to accelerate terminal differentiation, leading to acanthosis and epidermal hyperkeratosis phenotypes in mice and potentiate terminal differentiation of human keratinocytes in vitro 24,25 . Moreover, dioxin enhances differentiation of cells that have already committed to differentiation, insinuating that this environmental toxicant affects differentiation in a developmental stage-specific manner 26 .
Mouse embryonic stem cells (mESCs) are the inner cell mass cells isolated from the pre-implantation blastocysts 27,28 . When maintained under a well-defined culture condition, the mESCs have unlimited capacity of self-renewal, remain pluripotent with abundant expression of the pluripotency genes, such as Oct3/4 (Pou5f1) and Nanog 29 . The mESCs also have potent potential of differentiation to generate all the cell types of the body 30 . Under defined inducing conditions, they produce cells of the three primary germ layers, i.e. mesoderm, definitive endoderm and ectoderm, which in turn give rise to progenitor and mature cells of various lineages. Differentiation of mESC to generate epithelial cells for the purpose of tissue engineering and wound healing has been reported 31 , but it has not been used to explore the genetic and/or environmental factors as the underlying etiology of epithelial disorders.
In this study, we differentiated mESCs to basal keratinocytes in vitro. Retinoic acid (RA) and bone-morphogenetic protein-4 (BMP4) were used to induce early stage commitment of the surface ectodermal lineages; Defined keratinocyte serum free medium (DKSFM) were used to drive further differentiation to and the expansion of basal keratinocytes. The resultant basal keratinocytes, with epidermal progenitor signatures, could be passaged at least 20 times with minimal terminal differentiation. Using this system, we investigated the effects of the environmental toxicant dioxin and Map3k1 gene mutations, either individually or in combination, on epidermal differentiation. The results highlight the utility of the in vitro system to investigate risk factors and multifactorial etiology in congenital skin disorders without extensive utilization of live animals.

Results
In vitro differentiation of mouse ESCs to keratinocyte. We differentiated the mESCs in vitro using a protocol adapted from Bilousova et. al. and Metallo et al. with modifications to increase efficiency at the initial phase of the procedure 32,33 (Fig. 1A). Specifically, mESCs were re-suspended in EB media and the hanging drop methods were used for EB formation. Under these conditions, compact, sphere-shaped EBs formed in nearly 80% of the handing drops (Fig. 1B). After plating on ColIV-coated plates and growing in DKSFM media, the EBs The role of MAP3K1 in keratinocyte differentiation. MAP3K1 is a signal transduction enzyme playing key roles in embryonic development and epithelial morphogenesis 16 . Although the Map3k1 -/mice do not have overt skin defects, they display eye developmental defects due to abnormal epithelial morphogenesis and delayed healing of skin full-thickness wounds 17 . To explore the role of MAP3K1 in skin biology, we re-analyzed the global gene expression in wild type and Map3k1 -/primary keratinocytes 17 , using a more stringent cut-off criteria and performed GO analyses of the differentially expressed genes. We found that skin development was www.nature.com/scientificreports/ a top biological process affected by MAP3K1 (Fig. 3A). Specifically, genes in epithelial terminal differentiation were significantly up-regulated in Map3k1 -/versus wild type cells 17 (Supplementary Table s1).
To evaluate whether the roles of MAP3K1 in epithelial differentiation could be recapitulated in the in vitro differentiation model, we differentiated the wild type and Map3k1 -/-mESCs and examined the expression of marker genes at different time intervals. Wild type and Map3k1 -/cells had similar expression of Krt 18 at the early phase of differentiation (Fig. 3B). While these cells also had similar Krt 14 expression, they were strikingly different on Krt 1 expression at the later phase of differentiation (Fig. 3C). The expression levels of Krt 1 mRNA were tenfold more abundant in Map3k1 -/than in wild type cells. Similarly, there was a significant nearly twofold higher Krt 1 protein in Map3k1 -/than wild type cells ( Fig. 3D and E). To validate the differentiation status of the D-KC cells, we examined additional markers. Compared to the wild type cells, the Map3k1 knockout cells had a slightly higher expression of Krt 10 and Involucrin, markers for spinous and granular cells, respectively; however, they had similar expression of Loricrin and Filaggrin, which are markers for the most outer layer stratum corneum (Fig. 3F) 41 . The in vitro data validate that MAP3K1 hampers epithelial differentiation, an idea originally insinuated from global gene expression studies.

Dioxin potentiate keratinocyte differentiation in vitro.
The global environmental pollutant dioxin exhibits diverse developmental toxicities and is suggested to cause acanthosis and epidermal hyperkeratosis through derailing epithelial differentiation 24 . As most dioxin effects are mediated by the Aryl Hydrocarbon Receptor (AHR), a ligand-activated transcription factor that regulate dioxin-responsive genes 42 , we examined AHR expression during in vitro epithelial differentiation. The expression of Ahr was negligible in mESCs, but was significantly increased as soon as the cells started to differentiate in the EBs at day 2 of differentiation, consistent with previous observations 43 (Fig. 4A). The Ahr expression continuously increased and remained at high levels after the cells committed to primary ectodermal lineages and became basal keratinocytes, suggesting that AHR signaling could be activated by dioxin as soon as the cells exit stemness.
To evaluate the effects of dioxin on differentiation, we examined cells differentiated in media with or without dioxin for 13 days. The presence of dioxin in the culture media did not alter the expression of Krt 18 and Krt 14, suggesting that dioxin did not change the course of differentiation from mESC to surface ectoderm and basal keratinocytes (Fig. 4B). After multiple passages (> 8), the steady-state D-KC exhibited a robust dioxin-induced AHR activation, reflected by the induction of Cyp1a1, the prototypical AHR target gene (Fig. 4C). While the presence of dioxin did not change Krt 14 expression, it increased Krt 1 expression by threefold, although such increase was not detected at the protein level likely due to insufficient sensitivity of the detection methods (data www.nature.com/scientificreports/ not shown). In addition to Krt 1, Krt 10 and Involucrin mRNA were also slightly induced by dioxin treatment (Fig. 4D), supporting that dioxin treatment potentiates basal to spinous keratinocyte differentiation.

Dioxin plus Map3k1 loss-of-function further promote differentiation. In vivo, dioxin and
Map3k1 +/have synergistic effects on impairing eye development. When neither dioxin nor Map3k1 +/alone are detrimental, their combination causes birth defects of the eye, a defect observed also in un-treated Map3k1 -/mice 44 . Notwithstanding the intriguing phenotypic observations, how the environmental and genetic factors converge to disrupt the developmental programs has remained elusive. Given the similar effects of dioxin and Map3k1 gene mutation on promoting suprabasal differentiation, we postulated that the combination of these conditions exacerbated the differentiation abnormalities. In supporting of this idea, we noted that compared to the un-treated and dioxin-treated wild type cells, the dioxin-treated Map3k1 -/cells had also increased expression of Loricrin, another cornified envelop marker (Fig. 4D). We additionally tested the idea by treatment of D-KC derived from wild type and Map3k1 +/-mESCs with 10 nM dioxin for 3 days and examination of Krt 1 expression. Compared to the wild type cells, the Map3k1 +/cells had higher Krt 1 expression (Fig. 5A). We also tested this idea in vivo by treatment of pregnant mice, carrying wild type and Map3k1 +/embryos, with 50 ug/kg dioxin on embryonic day (E)11.5. The embryos were collected on E15.5, as described previously 44 and the embryonic skin was examined by immunohistochemistry. The E-cadherin staining labeled multiple layers of the epithelial cells in the embryonic skin, in which the Krt 1 positive cells were detectable at the most outer layer (Fig. 5B). Quantification of the signal intensities showed that neither dioxin exposure nor Map3k1 +/altered the level of Krt 1 expression; however, their combination significantly increased Krt 1 in more than 20 samples examined (Fig. 5C). The in vivo and in vitro data together raise an intriguing possibility that the geneenvironment interactions significantly potentiate basal to suprabasal differentiation as a potential mechanism underlying the eye developmental abnormalities.

Discussion
In this paper, we describe an experimental system that differentiates mESCs to basal keratinocytes in vitro. The system enables convenient incorporation of genetic and environmental components, leading to the findings that Map3k1 loss-of-function and dioxin, while do not affect mESC differentiation to surface ectoderm and basal keratinocytes, jointly potentiate basal to suprabasal epidermal differentiation. Compelled by these in vitro findings, we examined the gene-environment interactions in vivo and found that in utero dioxin exposure indeed increased Krt 1 expression more abundantly in the Map3k1 +/than in the wild type embryos. These data suggest that the in vitro system described here can be used to explore complex conditions and etiology in the perturbation of epithelial differentiation. Dioxin is a ubiquitous environmental agent that is stable and persistent in the environment and biological systems 45 . Consistent with the notion that most toxic effects of dioxin are mediated through the AHR, we found a good correlation between Ahr expression and dioxin effects on differentiation 46 . The minimal Ahr expression in mESCs and early phase of differentiation corresponded with unaltered differentiation from mESC to progenitor to basal keratinocytes in the presence of dioxin. The gradually increased Ahr and the steady-state high expression in the basal keratinocytes corresponded to potentiation of basal to spinous differentiation by dioxin. A similar observation has been made in the human cell culture models where dioxin is found to accelerate keratinocyte terminal differentiation, but does not change proliferation and apoptosis 25,26 . It is worth noting that of the many clinical manifestations of dioxin exposure, chloracne, a hyperkeratotic skin disorder is the most consistent pathology observed in exposed humans 47,48 . Thus, potentiation of basal to spinous differentiation observed here is likely relevant to dioxin-induced skin pathogenesis.
How dioxin accelerates differentiation remains unclear. Dioxin treatment of basal keratinocytes led to a significant up-regulation of Cyp1a1, an AHR-regulated detoxification gene. However, the detoxification gene www.nature.com/scientificreports/ products have not been causally linked to Krt 1 expression and differentiation. The dioxin-AHR axis also regulates expression of genes that are not implicated in detoxification, such as transforming growth factor-a, epidermal growth factor 49 , Interleukin-1b and Plasminogen activator inhibitor-2 50 . Some of these gene products may mediate the toxicities of dioxin. For example, in a mouse model of embryonic palate fusion, Abbott et. al. showed that dioxin induces epithelial differentiation abnormalities to cause cleft palate in wild type, but not Egf knockout mouse palates, implicating a role for EGF in dioxin toxicity 51 .
We have previously shown that MAP3K1 is a signaling molecule crucial for eye development and that the Map3k1 -/but not Map3k1 +/embryos have eyelid closure defects due to epithelial morphogenetic abnormalities 16 . More recently, we found that Map3k1 gene mutations sensitize the developmental programs to the toxicity of dioxin-like environmental chemicals 44 . Specifically, in utero dioxin exposure induces eye defects in Map3k1 +/but not wild type embryos. The in vitro mESC epithelial differentiation model, followed by in vivo validation, suggests that the gene (Map3k1)-environment (dioxin) interactions affect epithelial differentiation. The in vitro and in vivo data present a coherent narrative that Map3k1 mutation and dioxin have small effects on promoting basal to suprabasal differentiation, which is further potentiated by both agents together. The differentiation abnormalities induced by dioxin plus Map3k1 +/resemble those in Map3k1 -/cells, raising an intriguing possibility that the developmental defects occur when the differentiation abnormalities reaching beyond a threshold level.
Our differentiation protocol is modified from Metallo et. al. and Bilousova, et. al. with improved efficiency 32,33,52 . Using this protocol, we detected a gradual increase of Krt 18 expression, reaching the peak level in 10 days that are comparable to the time frame required for mouse stem cells to commit to ectodermal lineage in vivo 53 . We further obtained Krt 14-expressing cells after culturing for approximately a month, though this time frame was much longer than that took in vivo for ectodermal to basal epithelial cell conversion. Given that the Krt 18-positive to Krt 14-positive conversion requires DNA methylation to turn off the Krt 18 promotor and multiple extracellular signals and transcription factors to activate the Krt 14 promoter, we speculate that these epigenetic and transcription machineries are less robust in vitro than in vivo [54][55][56] .
In vivo, the basal layer epidermis has low Ca 2+ concentrations that support basal keratinocyte proliferation, whereas the suprabasal layers have high Ca 2+ concentrations to promote terminal differentiation. Additionally, the three-dimensional (3D) in vivo microenvironment facilitate the intricate cell-matrix interactions and differentiation gene expression 57 . The in vitro conditions, i.e. monolayer culture and the DKSFM media containing 0.15 mM calcium similar to the Ca levels in the basal layer epidermis, on the other hand, seem to favor basal keratinocyte proliferation but prevent differentiation 58,59 . Preliminary characterization shows that the D-KC, while lacking stem cell markers, are enriched with epidermal progenitors, explaining the continuously growth and subculture capacities of the Krt 14-positive cells.
In summary, we have established an experimental model that differentiates mESCs to keratinocytes that recapitulates epithelial differentiation from E3.5 to E15.5. This system can be used as a convenient tool to trace epithelial differentiation; the resultant basal keratinocytes can be amplified and cultured for a long period of time, serving as resources for epithelial molecular biology research. Using this system, we show that Map3k1 loss-offunction mutation and dioxin act jointly to potentiate epithelial spinous differentiation, unveiling a potential mechanism through which gene-environment interactions, but not each agent separately, cause developmental abnormalities. Understanding the complex etiology of diseases will help the development of preventive strategies. Mice, mESC culture and differentiation. The wild type and Map3k1 +/mice, in utero dioxin exposure and the collection and process of embryos for immunostaining were described before 44 . The wild type, Map3k1 +/and Map3k1 -/-mESCs were obtained from pregnant mice as described 60 . The mESCs were expanded and maintained in DMEM supplemented with 15% Knockout™ Serum Replacement (Gibco), LIF (10000x), 2 mM glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 2-mercaptoehanol (Gibco, 1000x), 100 U/ml penicillin and 100 μg/ml streptomycin (Cytiva), in a humidified incubator with 5% CO 2 at 37 °C. Protocol describing mouse experiments and procedures was approved by IACUC of the University of Cincinnati, and all experiments were performed in compliance with the ARRIVE guidelines and UC guidelines and regulations.

Reagents
The step-wise differentiation of mESCs to the epithelial lineages followed protocols in Bilousova, et al. 32 and Metallo, et al. 33 with modifications. Briefly, on day 0, mESCs were trypsinized and resuspended in DMEM with 15% fetal bovine serum (FBS), known as embryoid body (EB) media; cells (5 × 10 4 cells/25 µl) were placed as droplets on a Petri dish lid and incubated as "hanging drops" to enable the formation of EBs that contained cells of the three primitive germ layers 30 . On day 2, the EBs (about 100 in number) were collected and transferred to a 100 mm ColIV-coated tissue culture dish in EB medium plus 1 μM RA and 25 ng/ml BMP4, conditions that selectively induce surface ectoderm differentiation. On day 4, media were changed to DKSFM plus RA and BMP4 to promote epithelial lineage differentiation. On day 8, media were changed to DKSFM for keratinocyte amplification. On day 13, many cells with epithelial morphology were moving outward from the EB center. The clumps at the EB center was removed by vacuum aspiration; the remaining cells were detached with TrypLE, resuspended in DKSFM containing 10 mg/ml trypsin inhibitor and passaged to a new ColIV-coated dish, as passage (P) 1 Immunofluorescence, microscopic image and quantification. The embryonic tissue sections were processed and immunohistochemistry was done as described previously 16,61 . Briefly, the embryonic/fetal heads were fixed in 4% paraformaldehyde at 4 °C overnight. The tissues were embedded in Optimal Cutting Temperature compound and frozen. The entire eye was processed for coronal sections at 12 μM. Cells grown on ColIVcoated coverslips were fixed with 4% paraformaldehyde at 4 °C for 10 min, permeabilized with PBS plus 0.2% Triton, and subjected to immunofluorescent staining. Primary antibodies were diluted at 1:100 and secondary antibodies and nucleus staining reagents were dilute at 1:400. Immunofluorescence and bright field images were captured using a Zeiss Axio microscope. For immunostaining of the embryonic tissues, the imagens were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The epithelial cell layers expressing distinctive E-cadherin were outlined and the mean intensity values of Krt1 staining were measured. Krt1 level in the images was determined after background subtraction.
Global gene expression and pathway analyses. The wild-type and Map3k1 -/primary mouse keratinocytes were subjected to high-density microarray hybridization; the differential gene expression was analyzed as reported before 17 and are available at GSE201823. The data were re-analyzed through identifying the significantly differential expressed genes using the cut-off criteria: log2 fold change > 1 or < -1, False Discovery Rates (FDR) < 0.1, and intensity > 200, and the biological process enrichment using Metascape as previously described 62 . The datasets generated and/or analyzed during the current study are available.

Statistical analyses.
Means and standard deviations were calculated based on at least three independent experiments, and analyzed using student's two-tailed t-test. *p, # p < 0.05, **p < 0.01 and ***p, ### p < 0.001 were considered statistically significant.

Data availability
The datasets and cells generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.